NK cells provide first line surveillance against cells that have been infected by viruses or undergo neoplastic transformation. Among the numerous cell surface receptors mediating NK cell recognition of target cells, NKG2D is unique in that it recognizes a variety of class I-related molecules that serve as "flags" for infected or abnormal cells. In vitro studies indicate that NKG2D depends solely on the transmembrane adapter DAP10 for signaling. DAP10 contains an YxxM motif that recruits Phosphatidyl Inositol-3 Kinase. The same docking motif has been reported in CD28, which mediates a costimulatory signal in naive T cells. Therefore, NKG2D/DAP10 is currently considered a costimulatory complex on all NK cells as well as on activated cytotoxic T cells. This functional parallel between NKG2D/DAP10 and CD28 raises several important issues: Do NK cells require a costimulatory signal to kill their targets and/or proliferate in vivo? Is NKG2D/DAP10 involved in priming and expansion of CD8+ T cells in peripheral lymphoid organs, similar to CD28? Or, does NKG2D/DAP10 mainly enhance CTL effector responses against cells expressing NKG2D ligands in peripheral tissues? To address these crucial questions we have generated DAP10-deficient mice by gene targeting. Our preliminary results indicate that in NK cells, NKG2D has the capacity to associate not only with DAP10 but also with the adapter DAP12, which mediates direct activation via a distinct intracellular signaling pathway. In contrast, in T cells, NKG2D associates only with DAP10 and therefore is limited to the YxxM "costimulatory" pathway. On the basis of these results, we propose to dissect the role of NKG2D/DAP10 "costimulatory" and NKG2D/DAP12 "activating" pathways in NK cell biology by comparing NK cell function in DAP10 -/- mice with that of DAP12 -/- and normal mice in vitro and in vivo. In addition, we propose to clarify the role of the NKG2D/DAP10 "costimulatory" pathway in anti-viral and anti-tumor CD8+ T cell responses in vivo. For this purpose, we will analyse activation, expansion and acquisition of effector function of DAP10 -/- CD8+ T cell in mouse models of viral infection and tumor engraftment. In addition, we will study the requirement of DAP10 for establishment and maintenance of long-term memory CTLs following primary infections and tumor vaccination. While previous studies have indicated that expression of NKG2D ligands on virally infected and tumor cells is important for generating a protective immune response, it is not understood why and where NKG2D signaling is required during the response. Therefore, detailed knowledge of NKG2D/DAP10 function in vivo based on the analysis of DAP10-/- mice will be of great value, particularly in view of the possible exploitation of NKG2D-NKG2D-ligands interactions in tumor therapy and vaccine design.